Inflammasomes are cytosolic multiprotein complexes that sense microbial infection and trigger cytokine production and cell death. However, the molecular components of inflammasomes, and what they sense, remain poorly defined. Here we demonstrate that 35 amino acids from the Cterminus of flagellin triggered inflammasome activation in the absence of bacterial contaminants or secretion systems. To further elucidate the host flagellin-sensing pathway, we generated mice deficient in Naip5. Naip5-deficient mice failed to activate the inflammasome in response to the 35 amino acids of flagellin or in response to Legionella pneumophila infection. Taken together, these data clarify the molecular basis for the cytosolic response to flagellin.Inflammasomes are cytosolic multiprotein complexes that are critical regulators of inflammation, and are required for proteolytic activation of the cysteine protease caspase-1 (refs. 1-3). Caspase-1 (A000492; http://www.signaling-gateway.org/molecule/query?afcsid=A000492) is itself required for the proteolytic processing and release of inflammatory cytokines such as interleukin 1β (IL-1β) and IL-18, as well as for induction of a necrotic-like cell death called pyroptosis1-3. The molecular components and structures of inflammasomes remain poorly defined. It is believed that multiple distinct inflammasomes may exist, each containing a key scaffold protein of the NLR (nucleotide-binding domain, leucine-rich repeat) superfamily that confers specificity for particular microbial products. For example, NLR proteins of the NLRP1 family (also called NALP1) appear to activate the inflammasome in response to anthrax lethal toxin4 and bacterial muramyl dipeptide5. In contrast, the NLR protein NLRP3 (also called NALP3 or cryopyrin) has been proposed to sense a wide range of stimuli including bacterial RNA6, viral DNA7, uric acid crystals8, muramyl dipeptide9,10, nigericin11, amyloid-beta12, and other irritants13-16. There is at present no molecular explanation for how a single NLR protein can NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript be activated by all these microbial products and the precise molecular nature of what is sensed by any inflammasome remains undefined.The inflammasome containing the NLR protein IPAF (also called NLRC4) is one of the best characterized inflammasomes, and has been proposed by several groups to respond to the presence of flagellin in the cytosol17-19. Flagellin-deficient mutants of Salmonella typhimurium and Legionella pneumophila are defective in IPAF-dependent inflammasome activation, and flagellin, purified from or expressed in bacteria, triggers IPAF-dependent caspase-1 activation when delivered to the cytosol of macrophages by use of a pore-forming toxin (listeriolysin O (LLO)) or transfection reagents17-21. It was proposed that during natural infections, flagellin triggers inflammasome activation upon secretion into the host cytosol via bacterial type III/IV secretion systems17-21. However, doubts have been expressed as to w...

SummarySurvival and replication inside host cells by Brucella spp. requires a type IV secretion system (T4SS), encoded by the virB locus. However, the identity of the molecules secreted by the T4SS has remained elusive. We hypothesized that proteins translocated by the T4SS would be co-regulated with the virB operon. The LuxR family regulator VjbR, known to regulate virB, bound a fragment of the virB promoter containing an 18 bp palindromic motif (virB promoter box), showing that VjbR regulated the virB operon directly. To identify virB co-regulated genes, we searched the Brucella suis 1330 and B. abortus 2308 genomes for genes with an upstream virB promoter box. One hundred and forty-four promoters in the two genomes contained the virB promoter box, including those of fliC encoding flagellin and cgs encoding cyclic b-glucan synthetase. Thirteen of these proteins were tested for VirB-dependent translocation into macrophages using a b-lactamase reporter assay. This analysis resulted in the identification of the proteins encoded by BAB1_1652 (VceA) and BR1038/ BAB1_1058 (VceC) as novel protein substrates of the Brucella T4SS. VceC could also be translocated by the Legionella pneumophila Dot/Icm T4SS into host cells. Our results suggest that VjbR co-ordinates expression of the T4SS and at least two of its secreted substrates.

Bacterial flagella are filamentous appendages on the cell surface that mediate bacterial motility. The filament of the flagellum is composed of ϳ20,000 flagellin subunits (1). Flagellin monomers of many bacterial species function as pathogen-associated molecular patterns and can be detected by host cells using both surface-localized toll-like receptor 5 (TLR5) 2 and cytosolic Nod-like receptors (2-6). The interaction of flagellin with TLR5 at the cell surface has been characterized in detail, identifying the structural basis for the TLR5-flagellin interaction (7,8). TLR5 sensing has been shown to be important in Legionnaires disease, a respiratory tract infection caused by the bacterium Legionella pneumophila, since human TLR5 polymorphisms correlated with disease susceptibility (7,8). In the intestine, TLR5 localizes to the basolateral surface of enterocytes and to CD11c ϩ lamina propria dendritic cells (9, 10). In these locations, TLR5 is thought to be involved in sensing a breach of the intestinal mucosa by enteroinvasive pathogens, triggering an inflammatory response that limits systemic spread of the bacteria (11).In addition to sensing by TLRs, flagellin was recently shown to be sensed by two different Nod-like receptors, Ipaf and Birc1e (also known as Naip5) (2, 4 -6, 12). Ipaf transmits a proinflammatory signal in response to flagellin by activating the inflammasome, resulting in caspase 1-dependent activation of Il-1␤, which is accompanied by cell death (pyroptosis) (13-17). Birc1e/Naip5 signals in response to flagellin, restricting intracellular growth of L. pneumophila by antagonizing the ability of the bacterium to avoid fusion with lysosomes and form a replicative phagosome (18). Several lines of evidence suggest that pathogens residing in a membrane-bound compartment of the cell, such as L. pneumophila and S. Typhimurium (Salmonella enterica serotype Typhimurium), may release flagellins from their vacuole into the host cytosol, which triggers signaling via Ipaf and Birc1e. First, S. Typhimurium and L. pneumophila mutants lacking flagellin elicit reduced caspase activation, IL-1␤ secretion, and cytotoxicity in macrophages (2, 4 -6, 19). Second, mutants of S. Typhimurium lacking the SPI-1 T3SS and L. pneumophila mutants lacking a functional Type IV secretion system (T4SS) fail to trigger Ipaf-dependent signaling (4,5,19). Third, introduction of purified flagellins into the cytosol of macrophages using pore-forming toxins or detergents or by heterologous expression in Escherichia coli expressing listeriolysin O triggers caspase-1 activation and IL-1␤ activation by an Ipaf-dependent mechanism (2, 4 -6, 20).Although injection of effector proteins into host cells by the S. Typhimurium SPI-1 T3SS has been visualized microscopically (21, 22), it has not yet been demonstrated that in infected cells, bacteria are able to translocate flagellin from a membrane-bound compartment into the cytosol. In this report, we show that S. Typhimurium translocates flagellin into the cytosol of infected cells by a process t...

Panax ginseng C.A. Meyer is one of the most highly valued medicinal plants in the world. To analyze the transcriptome of P. ginseng and discover the genes involved in ginsenoside biosynthesis, cDNAs derived from the total RNA of 11-year-old, wood-grown P. ginseng roots were analyzed by 454 sequencing. A total of 217,529 high quality reads (expressed sequence tags, ESTs), with an average length of 409 bases, were generated from a one-quarter run to yield 31,741 unique sequences. The majority (20,198; 63.6%) of the unique sequences were annotated using BLAST similarity searches. A total of 16,810 and 16,577 unique sequences were assigned to functional classifications and biochemical pathways based on Gene Ontology analysis and the Kyoto Encyclopedia of Genes and Genomes assignment, respectively. Nine genes involved in the biosynthesis of ginsenoside skeletons and many candidate genes putatively responsible for modification of the skeletons, including 133 cytochrome P450s and 235 glycosyltransferases, were identified. From these candidates, six transcripts encoding UDP-glycosyltransferases that were most likely to be involved in ginsenoside biosynthesis were selected. These results open a new avenue by which to explore and exploit biosynthetic and biochemical properties that may lead to drug improvement. These 454 ESTs will provide the foundation for further functional genomic research into the traditional herb P. ginseng or its closely related species.

The Brucella abortus virB operon, encoding a type IV secretion system (T4SS), is required for intracellular replication and persistent infection in the mouse model. The products of the first two genes of the virB operon, virB1 and virB2, are predicted to be localized at the bacterial surface, where they could potentially interact with host cells. Studies to date have focused on characterization of transposon mutations in these genes, which are expected to exert polar effects on downstream genes in the operon. In order to determine whether VirB1 and VirB2 are required for the function of the T4SS apparatus, we constructed and characterized nonpolar deletion mutations of virB1 and virB2. Both mutants were shown to be nonpolar, as demonstrated by their ability to express the downstream gene virB5 during stationary phase of growth in vitro. Both VirB1 and VirB2 were essential for intracellular replication in J774 macrophages. The nonpolar virB2 mutant was unable to cause persistent infection in the mouse model, demonstrating the essential role of VirB2 in the function of the T4SS apparatus during infection. In contrast, the nonpolar virB1 mutant persisted at wild-type levels, showing that the function of VirB1 is dispensable in the mouse model of persistent infection.Brucella spp. are found in association with a large number of wild and domesticated animal species, where they can cause persistent infection and abortion. Zoonotic transmission of the organism to humans can lead to a chronic febrile disease known as brucellosis or Malta fever. A characteristic of both natural and zoonotic forms of the disease is extended survival of the organism in tissues of the reticuloendothelial system, such as the spleen, lymph nodes, and bone marrow.In a mutant screen for Brucella abortus virulence factors required for survival in the murine reticuloendothelial system, the virB operon, encoding homologs of Agrobacterium tumefaciens and Bordetella pertussis type IV secretion systems (24), was found to be essential for persistence in mice (18). The importance of these genes for intracellular survival was demonstrated in tissue culture models of infection as well (12,15,20,24,30). These genes were subsequently found to be expressed intracellularly by Brucella suis in macrophages (8), where they are required for localization of B. abortus to an intracellular niche that is associated with the endoplasmic reticulum (9), the same location where B. abortus was earlier shown to replicate in nonprofessional phagocytes (13,14,27) and in the ruminant placenta (2, 23). For B. abortus, the virB genes have been implicated in the initial interactions between the bacterium and the host cell during entry into macrophages (33).Based on studies performed with A. tumefaciens, both VirB1 and VirB2 are predicted to be accessible on the surface of B. abortus. VirB2 is predicted to form a pilus-like structure, and VirB1 is predicted to have two domains-a lytic transglycosylase and a second domain that is released to the extracellular medium and remains a...

Weak electric fields guide cell migration, known as galvanotaxis/electrotaxis. The sensor(s) cells use to detect the fields remain elusive. Here, we perform a large-scale screen using an RNAi library targeting ion transporters in human cells. We identify 18 genes that show either defective or increased galvanotaxis after knockdown. Knockdown of the KCNJ15 gene (encoding inwardly rectifying K+ channel Kir4.2) specifically abolishes galvanotaxis, without affecting basal motility and directional migration in a monolayer scratch assay. Depletion of cytoplasmic polyamines, highly positively charged small molecules that regulate Kir4.2 function, completely inhibits galvanotaxis, whereas increase of intracellular polyamines enhances galvanotaxis in a Kir4.2-dependent manner. Expression of a polyamine binding-defective mutant of KCNJ15 significantly decreases galvanotaxis. Knockdown or inhibition of KCNJ15 prevents PIP3 from distributing to the leading edge. Taken together these data suggest a previously unknown two-molecule sensing mechanism in which KCNJ15/Kir4.2 couples with polyamines in sensing weak electric fields.

A large number of hypothetical genes potentially encoding small proteins of unknown function are annotated in the Brucella abortus genome. Individual deletion of 30 of these genes identified four mutants, in BAB1_0355, BAB2_0726, BAB2_0470, and BAB2_0450 that were highly attenuated for infection. BAB2_0726, an YbgT-family protein located at the 3′ end of the cydAB genes encoding cytochrome bd ubiquinal oxidase, was designated cydX. A B. abortus cydX mutant lacked cytochrome bd oxidase activity, as shown by increased sensitivity to H2O2, decreased acid tolerance and increased resistance to killing by respiratory inhibitors. The C terminus, but not the N terminus, of CydX was located in the periplasm, suggesting that CydX is an integral cytoplasmic membrane protein. Phenotypic analysis of the cydX mutant, therefore, suggested that CydX is required for full function of cytochrome bd oxidase, possibly via regulation of its assembly or activity.

Many types of cells migrate directionally in direct current (DC) electric fields (EFs), a phenomenon termed galvanotaxis or electrotaxis. The directional sensing mechanisms responsible for this response to EFs, however, remain unknown. Exposing cells to an EF causes changes in plasma membrane potentials (V m ). Exploiting the ability of Dictyostelium cells to tolerate drastic V m changes, we investigated the role of V m in electrotaxis and, in parallel, in chemotaxis. We used three independent factors to control V m : extracellular pH, extracellular [K ؉ ], and electroporation. Changes in V m were monitored with microelectrode recording techniques. Depolarized V m was observed under acidic (pH 5.0) and alkaline (pH 9.0) conditions as well as under higher extracellular [K ؉ ] conditions. Electroporation permeabilized the cell membrane and significantly reduced the V m , which gradually recovered over 40 min. We then recorded the electrotactic behaviors of Dictyostelium cells with a defined V m using these three techniques. The directionality (directedness of electrotaxis) was quantified and compared to that of chemotaxis (chemotactic index). We found that a reduced V m significantly impaired electrotaxis without significantly affecting random motility or chemotaxis. We conclude that extracellular pH, [K ؉ ], and electroporation all significantly affected electrotaxis, which appeared to be mediated by the changes in V m . The initial directional sensing mechanisms for electrotaxis therefore differ from those of chemotaxis and may be mediated by changes in resting V m .Cells migrate directionally in response to many extracellular cues including chemical gradients (chemotaxis), topography, mechanical forces (mechanotaxis/durataxis), and electrical fields (EFs) (electrotaxis/galvanotaxis) (1,3,8,15,27). Electric fields have long been suggested to be a candidate directional signal for cell migration in development, wound healing, and regeneration. The mechanisms used by cells to sense the weak direct current (DC) EFs, however, have remained very poorly understood.One of the immediate effects felt by a cell upon exposure to an EF is a change in the cell membrane potentials (V m ). In an EF, the plasma membrane facing the cathode depolarizes while the membrane facing the anode hyperpolarizes (17, 18). It has been proposed that the changes in V m may underlie electrotaxis. In a cell with negligible voltage-gated conductance, the hyperpolarized membrane facing the anode attracts Ca 2ϩ by passive electrochemical diffusion. This side of the cell then contracts, thereby propelling the cell toward the cathode. In a cell with voltage-gated Ca 2ϩ channels, channels near the cathodal (depolarized) side open, thereby allowing Ca 2ϩ influx. Intracellular Ca 2ϩ levels will rise both on the anodal side and on the cathodal side in such a cell. The direction of cell movement in this situation will depend on the balance between the opposing contractile forces (17). The role of V m in electrotaxis has not yet been directly tested.In thi...